The differentiation, maturation, and maintenance of a complex tissue such as skeletal muscle requires ongoing cooperation between signaling pathways activated by hormones and growth factors, and intrinsic regulatory programs controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in normal muscle development in the fetus, are important for coordinating muscle regeneration following injury in the adult, and are critical for maintaining muscle mass during aging and in disease. The focus of this application will be on the IGF system in muscle development and repair, and represents part of a long-term effort to understand the mechanisms by which actions of the IGFs, their receptors, and binding proteins are integrated within the cell and in the whole organism. Based on current observations, we have postulated the existence of a robust autocrine signaling network in muscle cells that is critical for differentiation. The first key step in activating this network is production and secretion of IGF-II by differentiating myoblasts, which is secondary to induction of IGF-II gene transcription. Secreted IGF-II binds to and activates the IGF-I receptor, which leads to sustained stimulation of the PI3-kinase - Akt signaling pathway. Akt activity, principally Akt1, then plays a central role in driving early differentiation, in part by enhancing muscle gene expression through stabilization of active transcriptional complexes on muscle promoters. As differentiation proceeds, signal amplification occurs as more Akt2 is produced, and both Akts then participate in muscle cell maturation and in myocyte fusion to form multinucleated myotubes. I propose the following three Specific Aims to test this IGF-mediated signaling network hypothesis: 1. To determine the mechanisms of action of Akt1 in muscle differentiation. Proposed studies will test the idea that Akt1 is an obligate mediator of myoblast differentiation, and that its major functions are to enhance the actions of myogenic transcription factors on muscle gene promoters by stimulating key co-activators and inhibiting critical co-repressors. 2. To define the actions of Akt2 in muscle differentiation. Proposed experiments will examine the hypothesis that Akt2 acts to control late events in myoblast maturation and myotube formation. 3. To elucidate mechanisms of regulation of IGF-II gene transcription during muscle differentiation. Proposed experiments will test the hypothesis that a critical control region for differentiation-dependent activation of IGF-II gene transcription resides downstream of the H19 gene. Major goals will be to define the mechanisms of action of this chromosomal response element. PUBLIC HEALTH RELEVANCE: Insulin-like growth factors (IGFs) play key roles in normal muscle development in the fetus, are important for coordinating muscle regeneration following injury in the adult, and are critical for maintaining muscle mass during aging and in disease. The focus of this application will be on the IGF system in muscle development and repair, and represents part of a long-term effort to understand the mechanisms by which actions of the IGFs, their receptors, and binding proteins are integrated within the cell and in the whole organism.